1. The principle of laser confocal microscope
The traditional optical microscope uses a field light source, and the image of each point on the specimen will be interfered by the diffraction or scattered light of the adjacent point; the laser scanning confocal microscope (LSCM) uses a point light source to illuminate the sample, at the focal plane A small light spot with a clear outline is formed on the spot, and the fluorescent light emitted by this spot is collected by the objective lens and returned to the beam splitter composed of the dichromatic mirror along the original illumination light path. The spectroscope sends the fluorescence directly to the detector. There is a pinhole in front of the light source and the detector, which are respectively called the illumination pinhole and the detection pinhole. The illumination pinhole and the detection pinhole are conjugated with respect to the focal plane of the objective lens. The points on the focal plane focus on the illumination pinhole and the emission pinhole at the same time. The points outside the focal plane are blocked outside the detection pinhole and cannot be imaged. The obtained confocal image is the optical section of the specimen, which avoids the interference of stray light on the non-focal plane and overcomes the shortcomings of the ordinary microscope image blur, so it can obtain a clear confocal image on the entire focal plane.
Second, the principle of rotary disc confocal microscope
The ordinary microscope refers to a wide-field imaging microscope. The so-called wide-field imaging is "surface imaging". The so-called surface imaging is to obtain an entire two-dimensional image at a time point, which is obviously different from the scanning imaging microscope, such as a confocal microscope. Confocal microscopy is "point imaging", that is, only one spatial information can be obtained at each time point, and the entire 2D or 3D image is finally obtained by continuously spending time to scan, so each image point of scanning imaging is not a time point, regardless of scanning How fast is it. The current discs in the market are confocal, which is a combination of the two. Because porous discs can be used to obtain more space information at a time point, but the entire image is not obtained at a time point, and part of the spatial information is a time. Click to get.
Turntable disc confocal is multi-point synchronous scanning. Take Andromeda's turntable scanning head as an example. There are 20,000 spirally arranged pinholes on the turntable, and the laser light source covers about 2000 pinholes (that is, the scanning area). With the rotation of the turntable (the position of the pinhole changes accordingly), a complete scan of the sample is achieved, the sample in the scanning area is excited, the emitted light is conjugated with the excitation light irradiation point through the pinhole on the disc, and the non-focus plane stray light is filtered. It is equivalent to 2000 laser spots irradiating the sample synchronously, synchronous excitation, and synchronous acquisition on the CCD, so the scanning speed is very fast, and it is possible to obtain hundreds of thousands of confocal images per second. The biggest advantage of disc confocal is that the image quality can be close to or even reach the resolution of traditional confocal, at the same time, to obtain images of rapid changes in living cells.
3. Andromeda's disruptive optical design
Andromeda is a breakthrough improvement of the turntable confocal. The Spining disk turntable exclusively developed by Till is different from the previous double turntable disc type confocal. Only a turntable with a pinhole and a condenser lens is used to achieve both focusing and scanning. jobs. It provides a perfect multi-point disc confocal solution for three-dimensional imaging of continuous highly dynamic processes, such as cell division, protein transport or interaction, and encapsulation movement.
4. Application of laser scanning confocal microscope
(1) Three-dimensional reconstruction of cells
The resolution of the ordinary fluorescence microscope is low, and the displayed image structure is a multi-layer image overlay, and the structure is not clear enough. Laser confocal scanning can scan the cells in layers at 50nm steps along the axis to obtain a set of optical sections, which are stored as a two-dimensional array after A / D conversion. These arrays are processed by computers with different three-dimensional reconstruction algorithms, which can be used for monochrome or two-color image processing and combined into a real three-dimensional structure of the cell. Rotate different angles to observe the surface morphology of each side, and also observe the internal structure of the cell from different cross-sections, and measure the morphological parameters such as the length, width, height, volume and cross-sectional area of â€‹â€‹the cell. By simulating the fluorescent processing algorithm, the shadow effect formed at different lighting angles can be generated to highlight the three-dimensional feeling. Three-dimensional animation effects can be produced through angle rotation and cell position changes. The three-dimensional reconstruction of the laser confocal microscope is widely used in various types of cytoskeleton and morphology analysis, chromosome analysis, observation of programmed cell death, analysis and detection of structural changes in the cytoplasm and organelles of cells.
(2) Static structure detection: in situ identification of biological macromolecules in cells or tissues, observation of cell and subcellular morphological structure
1. In situ detection of nucleic acids in cells
It is used for nucleus location and morphology observation, detection of DNA replication and breakage in cells, and chromosome location observation.
2. Measuring proteins, antibodies and other molecules
Detection of proteins, antibodies and other molecules
Epidemic fluorescent labeling technology
Detect cell morphology and apoptosis-related proteins at different stages of apoptosis
4. Observation and determination
Probes can directly cross dead or live cell membranes, selectively binding to specific organelles
5. Cell fusion
6. Cytoskeleton structure
7. Intercellular gap connection communication
8. Detection of intracellular fat
Nile Red detects the content of lipid droplets in animal tissues or in vitro cultured cells
(3) Dynamic observation: real-time dynamic detection of the function of living cells or tissues
1. Real-time quantitative determination of changes in intracellular calcium
Calcium ion concentration determination and ratio imaging
2. Changes in intracellular pH
The same probe BCECF, using different excitation peaks to stimulate the fluorescence ratio to explore the PH changes
3. Detect changes in membrane potential
JC-1 monomer exists at low potential and emits green fluorescence; J-aggregate at high potential emits red fluorescence, and as the potential increases, red fluorescence also increases
4. Detect the generation of reactive oxygen species in cells
5. Drug screening
The process and location of transmembrane entry into tissues or cells
Viruses, bacteria and other fluorescent markers to detect entry, location content, dynamic process
6. Optical resonance energy transfer-FRET
GFP and YFP, BFP and GFP, etc. to study the interaction between macromolecules
7. Transportation Research
Using the endocytosis / exocytosis probe FM4-64, combined with laser confocal, electron microscopy, and TIRF, the vesicle movement process was detected to explore the effect of BFA on the endocytosis / exocytosis process.
8. Detection of fluorescent bleach recovery
The cells to be tested are labeled with a fluorescent substance and quenched; the low-intensity laser scans into a box, and the non-quenched molecular movement directly reflects the movement of the fluorescent labeled substance and its conjugate, so it can be used to study the speed of the gap connection communication.
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